Dengue virus infection is an arthropod-borne viral disease which has very high morbidity and mortality in humans. Dengue is a member of Flaviviridae, and utilizes an 11 kb single stranded, positive RNA genome. The genome encodes 3 proteins and contains additional non-coding regions at the 5' and 3' ends.
There are currently four different dengue virus subtypes known, which are distributed among geographically distinct tropical and subtropical regions. All four subtypes can cause an array of maladies, ranging from an acute, self limited illness (dengue fever, DF) to the more severe and potentially fatal dengue hemorrhagic fever or dengue shock syndrome (dengue hemorrhagic fever, DHF). As many as 50 million human cases occur annually, with an estimated 10,000 infant deaths due to the hemorrhagic form of dengue. Immunity to one serotype does not protect against infection by the others. In fact, sequential infection by another serotype substantially increases the probability of developing DHF (Rico-Hesse et al, Virology 230, 244-251 (1997)). For this reason it may be critical to be able to determine the genotype of the dengue virus for management of the disease.
Diagnosis and management of the disease, as well as vector surveillance and epidemiological studies, would be facilitated by a rapid and sensitive assay for the specific type of dengue involved. Extensive cross reactions among the flaviviruses and the existence of four distinct dengue virus serotypes makes serotype identification difficult. Currently, the most reliable method for identification involves isolation of the virus in a sensitive host followed by serotype identification using reference antisera or monoclonal antibodies. This method usually takes two or more weeks, however. Alternatively, a four-fold or greater increase in antibody titer by standard serological tests can be used, but generally requires paired samples. An ELISA for the detection of dengue virus-specific IgM in patient serum has also been used in diagnosis; however, this assay has been shown to be of very limited sensitivity.
As a consequence of the limitations of the above methods, several attempts have been made to devise RT-PCR based assays for the detection and genotyping of dengue virus infection (Henchal et al, Am. J. Trop. Med. Hyg., 45(4), 1991, pp. 418-428; Lanciotti et al, Journal of Clinical Microbiology, 30(3), March 1992, p. 545-551; Chungue et al, Journal of Medical Virology, 40:142-145,1993; Vorndam et al, Journal of Virological Methods, 48(1994) 237-244; and Sudiro et al, Am. J. Med. Hyg., 56(4), 1997, pp. 424-429). These assays have relied on alternative strategies, including universal or specific primer amplification and probe detection, differential nested amplification, and RFLP analysis of the PCR products. To date, isothermal amplification methods for dengue RNA have not been reported.